Membrane and droplet-interface bilayer systems and methods

ABSTRACT

Droplet-interface bilayer and lipid bilayer membrane compositions stabilized with an amphiphilic polymer are disclosed. Methods of making and using the compositions are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to US application number 62/187,951,filed Jul. 2, 2015, the disclosure of which is incorporated herein byreference in its entirety.

BACKGROUND

Screening novel small molecule drugs against human ion channels is ofutmost importance in ensuring the safety and efficacy of 21st centurytherapeutics. Ligand and voltage-gated ion channels making up more than15% of FDA-approved drug targets, Unwanted blockage or stimulation ofion channels by drugs has caused severe side effects and deaths. Severalblockbuster drugs were pulled from the market because of these sideeffects. There are at least two reasons to be concerned with thisproblem. First, patients cannot be guaranteed their safety when takingnew medicines. Second, not every person is sensitive to these sideeffects, so they are effectively being denied treatment when a drug ispulled.

Currently, screening ion channels for unwanted drug interactions is anexpensive and painstaking process. Though methods based on fluorescentplate readers have accelerated the screening process, electrophysiologyis still required to understand how drugs and channels interact. Patchclamp electrophysiology requires live cells and sophisticatedinstrumentation, and a new cell line must be created for each channel ormutation to be studied. Reconstitution of channel proteins intoartificial membranes (lipid bilayers) can be performed, however thepurification and reconstitution of membrane proteins into lipid bilayersis an expensive, laborious and difficult practice.

U.S. Pat. No. 8,268,627 discloses a membrane system, thedroplet-interface bilayer, as an alternative technology to cell-basedmethods or planar bilayer methods for study of membrane proteinbehavior. Briefly, a replica cell membrane is created by joining twoindependently-formed lipid monolayers together. Two aqueous dropletscontaining lipid vesicles are submerged under an oily hydrocarbon,typically hexadecane. The lipid vesicles fuse at the oil/water boundaryof each droplet to form a self-assembled lipid monolayer around eachaqueous droplet. When the two droplets are brought into contact, thehexadecane is squeezed out from between the monolayers to create adroplet-interface bilayer (DIB). Membrane proteins present within one ofthe aqueous droplets insert into the bilayer. An Ag/AgCl electrodewithin each droplet enables the application of a voltage and themeasurement of ionic current flowing through channels in the DIB.

In vitro transcription and translation (IVTT) is a cell-free approachfor the synthesis of proteins from DNA templates. Many IVTT systems andin vitro transcription (IVT) products that synthesize proteins frommessenger RNA are now commercially available from a number of vendorsand are capable of producing integral membrane proteins such as ionchannels.

The cost of producing ion channels via IVTT reactions is relatively highwhen used with planar bilayer systems, where the aqueous compartmentvolumes are 100-1000 μL. Droplet-interface bilayers have emerged as asystem with greater potential for this application because the requiredvolumes are much lower (e.g., 200 nL per droplet) and the bilayers havehigher stability.

Bacterial and viral membrane proteins with both α-helical and β-barrelstructures, such as staphylococcal α-hemolysin, the potassium channelKcv from chlorella virus, and the potassium channel KcsA fromStreptomyces lividans, have been expressed by IVTT systems derived fromE. coli and then incorporated in DIBs.

In vitro expression of eukaryotic membrane proteins presents uniquechallenges. In eukaryotic organisms, synthesis of membrane proteinsproceeds in a series of steps that ensure proper folding andorientation. Much of a cell's interior is occupied by a network ofmembranes called the endoplasmic reticulum (ER). The ER contains ahighly-specialized docking site capable of threading secretory proteinsor inserting transmembrane protein segments into the lipid membrane.Called the translocon, this complex is believed to guide the channel'sinsertion such that only one orientation is possible. The insertion ofmembrane proteins into the ER is coupled to the protein's synthesis atthe ribosome. When the first segment of protein emerges from theribosome, it is bound by another protein complex called the signalrecognition particle (SRP). The SRP binds to both the nascent peptidechain and the ribosome, thereby pausing protein synthesis. The ERcontains a membrane-bound SRP receptor, which binds theSRP-peptide-ribosome complex. This membrane association effectivelydocks the ribosome with the translocon. When this occurs, the SRP isreleased and protein synthesis resumes, with each transmembrane domainthreading into the membrane one at a time. For membrane proteins, likeion channels, the first transmembrane a-helix is the binding site forSRP. In short, the ribosome, SRP, SRP receptor, and translocon worktogether to insert and orient membrane proteins such as ion channels.

In many cases, bacterial IVTT systems are ill-suited for eukaryoticmembrane protein expression within the DIB. Bacterial IVTT systems donot post-translationally modify the expressed proteins. Moreimportantly, the translocon is absent from such bacterial systems. To begenerally useful for animal or human membrane protein analysis, theIVTT-DIB approach would require all the components that eukaryoticorganisms use for membrane protein expression.

To synthesize ion channels within a DIB system, an in vitrotranscription/translation (IVTT) extract can be mixed with lipidvesicles and DNA for the desired membrane protein in the aqueoussolution of one of the precursor droplets. Including an E. coli IVTTsystem in a DIB, membrane proteins such as the viral potassium channelprotein (Kcv,) have been synthesized in situ. Unfortunately, thesebilayer interfaces were found to be very unstable, lasting only a coupleminutes. DIBs are even less stable when attempting to use any of avariety of eukaryotic expression systems, including rabbit reticulocytelysate, wheat germ extract, and yeast extract.

There is a need in the art for new compositions and methods forscreening ion channel-drug interactions that don't require cells ortraditional patch clamping techniques. Further, there is a need in theart for improved cell-free methods to analyze eukaryotic membraneprotein function.

SUMMARY

Disclosed herein are compositions for expression and analysis ofmembrane proteins in a droplet-interface bilayer and lipid bilayermembrane compositions.

In one aspect, a pair of droplets in a hydrophobic medium comprises afirst droplet of a first aqueous solution in the hydrophobic medium, thefirst droplet comprising a layer of amphipathic molecules around thesurface of the first aqueous solution, and containing atranscription/translation extract and a heterologous polynucleotideencoding a membrane polypeptide; and a second droplet of a secondaqueous solution in the hydrophobic medium, the second dropletcomprising a layer of amphipathic molecules around the surface of thesecond aqueous solution; the first droplet and the second droplet beingin contact with one another such that a bilayer of the amphipathicmolecules is formed as an interface therebetween; a polymer andoptionally the encoded membrane polypeptide are inserted into thebilayer.

In another aspect, a system comprises a bilayer of amphipathic moleculesprovided at the interface between a first droplet of a first aqueoussolution in a hydrophobic medium, the first droplet comprising a layerof amphipathic molecules around the surface of the first aqueoussolution, and containing a transcription/translation extract and aheterologous polynucleotide encoding a membrane polypeptide; and asecond droplet of a second aqueous solution in the hydrophobic medium,the second droplet comprising a layer of amphipathic molecules aroundthe surface of the second aqueous solution; wherein the bilayer containsa polymer and optionally the encoded membrane polypeptide.

In another aspect, a droplet of aqueous solution in a hydrophobic mediumcomprises a layer of amphipathic molecules around the surface of theaqueous solution, and containing a transcription/translation extract;and a heterologous polynucleotide encoding a membrane polypeptide andthe hydrophobic medium or an aqueous phase containing a polymer capableof insertion into a bilayer of the amphipathic molecules.

In another aspect, a system comprises a membrane separating first andsecond volumes of aqueous solution, the membrane comprising a lipidbilayer and an ion channel providing a passageway from one side of themembrane to the other, wherein the membrane contains 30% or less byweight of an amphiphilic polymer.

In another aspect, a membrane bilayer composition comprises a membranecomprising a lipid bilayer and an ion channel providing a passagewayfrom one side of the membrane to the other wherein the membrane contains30% or less by weight of an amphiphilic polymer.

In another aspect, a system comprises a membrane separating first andsecond volumes of aqueous solution, the membrane comprising a lipidbilayer and an ion channel providing a passageway from one side of themembrane to the other, wherein the first and/or second volumes ofaqueous solution comprises a membrane destabilizing agent and whereinthe membrane contains an amount of amphiphilic polymer effective forstabilization of the membrane.

In another aspect, a composition comprises a pair of droplets in ahydrophobic medium, the pair of droplets comprises a first droplet of afirst aqueous solution in the hydrophobic medium, the first dropletcomprising a layer of lipid molecules around the surface of the firstaqueous solution; and a second droplet of a second aqueous solution inthe hydrophobic medium, the second droplet comprising a layer of lipidmolecules around the surface of the second aqueous solution; the firstdroplet and the second droplet being in contact with one another suchthat a bilayer of the lipid molecules is formed as an interfacetherebetween; wherein the bilayer comprises an amount of amphipathicpolymer effective for stabilization of the bilayer.

In another aspect, a composition comprises a droplet in a hydrophobicmedium containing an aqueous solution and a hydrophilic layer, thedroplet comprising a layer of lipid molecules around the surface of thefirst aqueous solution and the hydrophilic layer comprising a layer oflipid molecules on the surface of the second aqueous solution; the firstdroplet and the hydrophilic layer being in contact with one another suchthat a bilayer of the lipid molecules is formed as an interfacetherebetween; wherein the bilayer contains an amount of amphipathicpolymer effective for stabilization of the bilayer.

Methods of making and using the compositions are also disclosed.

In one aspect, a method of forming a DIB system comprising a bilayer ofamphiphilic molecules provided at the interface between two droplets,comprises: forming a first droplet of a first aqueous solution in ahydrophobic medium, the first droplet comprising a layer of amphipathicmolecules around the surface of the first aqueous solution, andcontaining a transcription/translation extract; and a heterologouspolynucleotide encoding a membrane polypeptide; forming a second dropletof a second aqueous solution in the hydrophobic medium, the seconddroplet comprising a layer of amphipathic molecules around the surfaceof the second aqueous solution, bringing the droplets into contact withone another in the hydrophobic medium so that a bilayer of theamphipathic molecules is formed as an interface between the contactingdroplets; and incorporating a polymer into the bilayer.

In another aspect, a method of analyzing membrane polypeptide functioncomprises contacting a test compound with the DIB system or the membranesystem disclosed herein; and measuring a detectable signal from thesystem in the presence and in the absence of the test compound.

These and other embodiments, advantages and features of the inventionbecome clear when detailed description and examples are provided insubsequent sections.

BRIEF DESCRIPTION OF THE DRAWINGS

Referring now to the figures, which are exemplary embodiments, andwherein the like elements are numbered alike:

FIG. 1 is a drawing illustrating an exemplary DIB system for coupled invitro transcription/translation (IVTT)-channel analysis in which theleft droplet contains a eukaryotic transcription/translation extract anda plasmid encoding an ion channel gene which is expressed within thedroplet by the IVTT system, with subsequent insertion of the ion channelinto the bilayer and the right droplet contains molecules of a drug tobe tested for its effect on the ion channel.

FIG. 2 is a graph of an electrophysiology recording of activity as afunction of time of the pore-forming toxin a-hemolysin within apolymer-stabilized DIB containing a eukaryotic cell extract in onedroplet and the reversible pore blocker cyclodextrin in the otherdroplet. The inset shows a small section of the recording (black bar)expanded to show the reversible blockades by cyclodextrin.

FIGS. 3A, 3B, 3C, and 3D present expanded graphs of regions of FIG. 2marked with black triangles showing current spikes observed in thepolymer-stabilized IVTT-DIBs.

FIG. 4 presents a graph of an electrophysiology recording of activity asa function of time of the human kv11.1 channel (hERG gene) expressed insitu by an IVTT-containing DIB in the presence of a stabilizing polymer.The total recording time for this channel was over 40 minutes.

FIG. 5 presents a graph of an electrophysiology recording of toxin(α-hemolysin) activity in a DIB containing a eukaryotic cell extract inthe absence of stabilizing polymer. Signals were filtered with a 200 Hzlowpass Bessel filter.

FIG. 6 presents a graph of an electrophysiology recording of a DIBcontaining a eukaryotic cell extract and α-hemolysin as in FIG. 5,however the droplets of the DIB are equilibrated to a lower temperature(solutions on ice) before injection onto the electrodes. Beforeincorporation of the toxin, the two aqueous droplets coalescence withoutany sign of current leakage.

FIG. 7 presents a graph of an electrophysiology recording of a DIB,containing a eukaryotic cell extract and a-hemolysin, formed in ahydrophobic medium containing 0.05 g/L of triblock copolymer dissolvedin hexadecane; all other experimental conditions are the same as for theexperiment of FIG. 2.

FIG. 8 presents a graph of an electrophysiology recording for a dropletinterface bilayer, made by the rolling droplet approach, in the presenceof TBCP and a vast number of pores incorporated into the bilayer(α-hemolysin concentration approaching 1 mg/mL). In the early momentsonly one single pore is active (bottom left), after about 10 minutesother pores are being incorporated (Bottom right). After 10 minutes,droplets were detached to investigate the bilayer reforming (the firstarrow). After 27 minutes the presence of bilayer was checked throughcapacitance (second arrow). From minute 40 to 70 minutes, the currentwas stable around 1800 pA. After 70 minutes the voltage was switched to−70 mV to check the bilayer stability in both voltages.

FIG. 9 presents graphs of electrophysiology recordings for a dropletinterface bilayer, made by the rolling droplet approach in the presenceof TBCP and a large number of pores incorporated into the bilayer(α-hemolysin concentrations approaching 1 mg/mL. In fact, pores insertedinto the DIB until the conductance of the membrane exceeded the currentcapacity of the recording amplifier (at about 60 minutes, FIG. 9). Eventhough the current limit was reached, the DIB was still present for theentire recording as verified by visual inspection. Specifically, aruptured DIB would coalesce into one droplet whereas a stable DIB willstill be a pair of contacting droplets. A conventional lipid DIBmembrane would typically have ruptured at the insertion of so manypores. Inset panels in FIG. 9 are expanded regions of the hour-longrecording that show the reversible binding between cyclodextrin andhemolysin. These bindings are specific to hemolysin pores and do notoccur at membrane defects. Thus, all conductance of the DIB was due toions flowing through inserted hemolysin pores.

FIG. 10, panels a) and b) show the ability to inject a volume into apolymer-stabilized DIB. FIG. 10 shows a time interval of 2 secondsbetween panels a) and b). A lipid only DIB tends to rupture if a volumegreater that 10% of the droplet's volume is added. However, apolymer-stabilized DIB droplet could double in volume by pipetteinjection without rupturing. This provides the ability to add reagentsto a pre-formed DIB which would be useful in many types of tests.

FIG. 11 presents a schematic representation of the two approaches thathave been used to prepare a stabilized and robust Droplet InterfaceBilayer (DIB) using a tri-block copolymer. Panel (a) is a schematicrepresentation of the rolling approach; in which droplets are rolled (upand down and side-to-side) on a glass slide coated with a polymer-lipidmixture. The photographic inset shows the surface of a coated glasscoverslip before insertion into the oil bath. Panel (b) depicts thepolymer-in-oil bath approach. Briefly, the working electrode (left)contains buffer solution mixed with a dilute solution of the toxin(shown in the magnified cartoon is the α-hemolysin heptamer, RCSBProtein Data Bank structure 3ANZ). The ground electrode (right) containsbuffer solution and a human-derived in vitro transcription/translation(IVTT) extract of HeLa cell lysate with the desired concentration ofγ-cyclodextrin used to block the toxin activity.

DETAILED DESCRIPTION

A stable droplet-interface bilayer (DIB) system for coupled expressionand analysis of membrane proteins is disclosed herein. Previous DIBstructures for expression of membrane proteins including an in vitrotranscription/translation (IVTT) system within one of the droplets werefound to be highly destabilized compared to DIBs without the IVTTsystem, lasting only a few minutes. Complex IVTT extracts may lead toDIB instability for one or more reasons including imbalance of osmoticpressure between the droplets, detergents or other proprietary chemicalsadded to the matrix, high protein concentrations, or possiblybiochemical activity within the matrix.

Further disclosed herein is a membrane system comprising an ion channelthat has improved stability. The system may be used in the detection orcharacterization of an analyte. The system may be used in the deliveryof an analyte across the membrane through the ion channel. The systemmay comprise part of an interconnected droplet network.

The inventors have unexpectedly found that incorporating a small amountof stabilizing amphipathic polymer into the bilayer of anIVTT-containing DIB system stabilizes the DIB for at least 30 minuteswithout the two droplets coalescing. The disclosed IVTT-containing DIBsystems are stable for a sufficiently long time period to permitsynthesis of membrane proteins in situ within the IVTT-containingdroplet, insertion of the membrane protein into the bilayer of the DIB,and experimental analysis of the protein function. In particular, thedisclosed DIB systems permit electrophysiological recordings to detectmembrane protein function for extended time periods, producing nocurrent leakage or drift throughout the recording time period.Compositions and methods of making and using the DIB expression/analysissystem are disclosed.

Furthermore the inventors have unexpectedly found that the addition ofsmall amounts of amphipathic polymer to a lipid bilayer provideschemical stability and is able to make the lipid bilayer more robust andless susceptible to degradation by destabilizing agents. A lipid bilayermay rupture when exposed to such agents, including certain amphiphiles.Detergents and surfactants, including those commonly used to solubilizemembrane proteins, can cause defect formation and bilayer rupture evenat relatively low concentrations. Additives used to stabilizecell-derived expression extracts, such as glycerol and polyethyleneglycol (PEG), are also known to break DIB membranes. Components ofcell-derived expression extracts, including native fatty acids, lipids,proteins and other natural amphiphiles, along with additives, create amilieu that is highly destabilizing to lipid bilayer membranes. Highconcentrations of detergent-solubilized protein pores also tend to breaklipid bilayer membranes such as those in DIBs. However, lipid bilayerscontaining polymer amphiphiles have been shown to have an increasedresistance to rupture when exposed to such destabilizing agents. Thus,amphiphilic polymers such as those that protect DIBs from rupture impartchemical stability to lipid bilayers. The fact that only small amountsof amphipathic polymer were required in order to impart membranestability was surprising and provides a membrane which has the benefitsof both a lipid bilayer membrane and an amphipathic polymer membrane.

The addition of small amounts of amphipathic polymer to a lipid bilayermay also provide mechanical stability. Lipid bilayers are typicallysensitive to mechanical perturbation. Increasing the volume of onedroplet of a DIB (for example, by injection) may quickly lead torupture. Furthermore, the application of transmembrane voltages higherthan 150 mV typically cause lipid DIB droplets to coalesce. Forcesarising from osmotic imbalance, voltage, gravity, fluid flow, andmonolayer stretching due to droplet inflation all tend to destroy lipidbilayer membranes. However, when these forces are combined in thepresence of a polymer amphiphile-stabilized lipid bilayer membrane suchas a DIB, the membrane is stable for at least one hour. For example onedroplet of a stabilized DIB could be repeatedly injected with aqueoussolution without rupture (see FIG. 10 below) without the need toosmotically balance the droplets. In addition, defects that form inthose polymer amphiphile-stabilized DIBs appear to quickly self-heal(see FIG. 3 below). This is not observed in conventional non stabilizedlipid bilayer membranes.

In an embodiment the aqueous solutions may contain electrodes to providea potential difference across the membrane. Charged analytes such aspolynucleotides can be induced to translocate the ion channel duringwhich ion current measurements indicative of the analyte may be taken.The aqueous solutions would typically comprise an electrolyte. Theelectrodes may be reference electrodes such as AgCl. Alternatively thesolutions may comprise a suitable redox couple such asferri/ferrocyanide and the electrodes may be chosen from a relativelyinert material such as gold or platinum.

The first and/or second aqueous solutions may comprise an IVTT extract.

In an embodiment the system may further comprise one of more droplets,each droplet comprising a layer of lipid around its surface and formingan interconnected droplet network, wherein a lipid bilayer is formed ateach interface between droplets and wherein each lipid bilayer comprisesan ion channel. Such suitable systems are disclosed in WO2013/064837 andWO2014/064461, incorporated herein by reference.

Lipid bilayers comprising an ion channel are well known. The lipidbilayer disclosed herein may be prepared for example according to themethod of Montal & Mueller (Proc Natl Acad Sci USA. 1972 Dec.;69(12):3561-6). The lipid bilayer may be formed as a droplet interfacebilayer (DIB) or as an interface between a droplet and a planar surface,see for example WO2008/012552 and WO2009/024775 incorporated herein byreference. The may be an interconnected droplet network comprising threeor more droplets. The system disclosed herein may be used for examplefor the delivery of an analyte from one aqueous medium to the otherthrough the ion channel. The analyte may be a drug to be deliveredthrough the ion channel. The system may be used to detect orcharacterize an analyte , wherein the analyte is caused to translocatethe ion channel during which measurements are made, such as themeasurement of ion current flow under an applied potential difference,see for example WO2001/42782. The system may comprise an array ofmembranes for detection of analytes, each membrane comprising an ionchannel, such as disclosed by WO2009/077734. The analyte may be apolymer such as a polynucleotide or a polypeptide.

The use of a lipid bilayer membrane to incorporate an ion channel isideal in many respects in that it mimics the lipid membranes of cellwalls in which ion channels may be naturally present. However a drawbackof using lipid membranes is that they are fragile and may be subject todegradation which can result in damage to the membrane. Such damage mayresult in the lowering of the membrane resistance due to the presence ofleakage pathways or result in bursting In order to address this problem,non-lipid amphipathic polymer membranes have been developed into whichion channels may be incorporated, such as for example diblock andtriblock copolymers. An example of such ispoly(dimethylsiloxane)-block-poly(2-methyloxazoline) copolymer. Thesemembranes, being synthetic, are typically more resistant to degradationand have been shown to be more robust than lipid bilayer membranes whensubjected to high potential differences. However such membranes do notprovide a natural environment for insertion of an ion channel and theaddition of a detergent is often necessary in order to facilitate ionchannel insertion.

The aqueous solution may for example comprise a biological fluid or acell extract. The biological fluid may be blood, interstitial fluid,serum, urine, tears, saliva, or plasma. The aqueous solution may besemi-solid or comprise an extract from a solid sample.

Disclosed herein is a droplet-interface bilayer composition.

A droplet-interface bilayer (DIB) may be formed between two dropletswherein each droplet has a layer of amphipathic molecules around itssurface.

A droplet-interface bilayer may be formed between a droplet and ahydrophilic layer wherein the droplet has a layer of amphipathicmolecules around its surface and the hydrophilic layer has a layer ofamphiphilic molecules on its surface. The hydrophilic layer may beprovided on a solid support. The hydrophilic layer may be a hydrogel.The hydrophilic layer may be a hydrated support. Suitable examples ofsuch a droplet-interface bilayer are disclosed in WO2009/024775, hereinincorporated by reference.

Examples of destabilizing agents that can rupture or provide leakagepathways through otherwise highly resistive lipid bilayers includedetergents, surfactants, including those commonly used to solubilizemembrane proteins can cause defect formation and bilayer rupture even atrelatively low concentrations, additives used to stabilize cell-derivedexpression extracts such as glycerol and polyethylene glycol (PEG), andcomponents of cell-derived expression extracts, including native fattyacids, lipids, proteins and other natural amphiphiles.

In an embodiment, the composition comprises a pair of droplets in ahydrophobic medium. The pair of droplets comprises a first droplet of afirst aqueous solution in the hydrophobic medium, the first dropletcomprising a layer of amphipathic molecules around the surface of thefirst aqueous solution, and containing a transcription/translationextract and a heterologous polynucleotide encoding a membranepolypeptide; and a second droplet of a second aqueous solution in thehydrophobic medium, the second droplet comprising a layer of amphipathicmolecules around the surface of the second aqueous solution; the firstdroplet and the second droplet being in contact with one another suchthat a bilayer of the amphipathic molecules is formed as an interfacetherebetween; a polymer and optionally the encoded membrane polypeptideare inserted into the bilayer.

In an embodiment, the composition comprises a system comprising abilayer of amphipathic molecules provided at the interface between afirst droplet of a first aqueous solution in a hydrophobic medium, thefirst droplet comprising a layer of amphipathic molecules around thesurface of the first aqueous solution, and containing atranscription/translation extract and a heterologous polynucleotideencoding a membrane polypeptide; and a second droplet of a secondaqueous solution in the hydrophobic medium, the second dropletcomprising a layer of amphipathic molecules around the surface of thesecond aqueous solution; wherein the bilayer contains a polymer andoptionally the encoded membrane polypeptide.

In an embodiment, the composition comprises a pair of droplets in ahydrophobic medium. The pair of droplets comprises a first droplet of afirst aqueous solution in the hydrophobic medium, the first dropletcomprising a layer of lipid molecules around the surface of the firstaqueous solution, and containing a lipid bilayer destabilizing agent;and a second droplet of a second aqueous solution in the hydrophobicmedium, the second droplet comprising a layer of lipid molecules aroundthe surface of the second aqueous solution; the first droplet and thesecond droplet being in contact with one another such that a bilayer ofthe lipid molecules is formed as an interface therebetween; wherein thebilayer comprises an amount of amphipathic polymer effective forstabilization of the bilayer.

In an embodiment, the composition comprises a system comprising abilayer of lipid molecules provided at the interface between a firstdroplet of a first aqueous solution in a hydrophobic medium, the firstdroplet comprising a layer of lipid molecules around the surface of thefirst aqueous solution, and a second droplet of a second aqueoussolution in the hydrophobic medium, the second droplet comprising alayer of lipid molecules around the surface of the second aqueoussolution; wherein the bilayer contains an amphipathic polymer at amountof 30% by weight or less of the lipid molecules.

In an embodiment, the composition comprises a droplet in a hydrophobicmedium containing an aqueous solution and a hydrophilic layer, thedroplet comprising a layer of lipid molecules around the surface of thefirst aqueous solution and the hydrophilic layer comprising a layer oflipid molecules on the surface of the second aqueous solution; the firstdroplet and the hydrophilic layer being in contact with one another suchthat a bilayer of the lipid molecules is formed as an interfacetherebetween; wherein the bilayer contains an amphipathic polymer.

In an embodiment the aqueous droplet or the hydrophilic layer maycomprise a membrane destabilizing agent, in which case the amount ofamphipathic polymer. present in the bilayer is sufficient to stabilizethe bilayer. The aqueous droplet or the hydrophilic layer may comprise atranscription/translation extract and a heterologous polynucleotideencoding a membrane polypeptide.

The hydrophilic layer may be provided on a solid support. Thehydrophilic layer may be a hydrogel. The hydrophilic layer may be ahydrated support. Suitable examples of such a droplet-interface bilayerare disclosed in WO2009/024775, hereby incorporated by reference.

In an embodiment the membrane contains 30% or less by weight of anamphiphilic polymer, such as 25%, 20%, 15% or less.

In an embodiment the membrane contains, between 0.1% and 10% by weightof an amphiphilic polymer, such as 0.2%, 0.5%, 1%, 5%, or more.

In an embodiment the first and/or second volumes of aqueous solutioncomprise a lipid bilayer destabilizing agent and the membrane or DIBcontains an amount of an amphiphilic polymer effective forstabilization.

The first or second volumes of aqueous solution may comprise atranscription/translation extract and a heterologous polynucleotideencoding a membrane polypeptide.

The disclosed compositions, systems, pair of droplets or thedroplet-interface bilayer system can be used in assays to analyzeexpression and/or function of a membrane protein inserted into thebilayer interface between the two droplets.

Also disclosed herein is a droplet of aqueous solution in a hydrophobicmedium, the droplet comprising a layer of amphipathic molecules aroundthe surface of the aqueous solution, and containing atranscription/translation extract; and a heterologous polynucleotideencoding a membrane polypeptide and the hydrophobic medium or an aqueousphase containing a polymer capable of insertion into a bilayer of theamphipathic molecules.

The disclosed droplet can be used to prepare a droplet-interface bilayersystem for coupled expression and analysis of the encoded membranepolypeptide.

In any of the disclosed compositions, the encoded membrane polypeptidecan be a channel or a pore. The encoded membrane polypeptide can be aprokaryotic or eukaryotic polypeptide.

The phrases “in vitro transcription/translation (IVTT)” system and“transcription/translation” system are used interchangeably herein andmean a cell-free system for the synthesis of proteins from DNAtemplates. Usually, a combination of cell extracts and purifiedcomponents are combined from multiple sources and optimized to produceeither soluble or membrane proteins. Commercially available cell-freeprotein synthesis systems are typically derived from cell extracts ofEscherichia coli S 30 , rabbit reticulocytes, or wheat germ. Thedrawback of extract-based systems is that they often contain nonspecificnucleases and proteases that adversely affect protein synthesis.Additionally, the first IVTT system formulated from individuallypurified components from E. coli was developed in 2001 and called the“protein synthesis using recombinant elements” or PURE system (Shimizu Aet al. Nat Biotechnol 2001, 19:751- 755 ). The transcription/translationsystem in the disclosed compositions and methods can be a eukaryotictranscription/translation system. In some embodiments, the eukaryotictranscription/translation system comprises a eukaryotic cell extract. Anumber of eukaryotic cell-free protein expression systems have beenderived from yeast, rabbit reticulocytes, wheat germ, insects, andimmortalized human cell lines, for example Hela or HEK cells. EukaryoticIVTT systems are commercially available, e.g., a HeLa IVTT system(ThermoFischerScientific Cat. No. 88882), however noncommercial systemscan also be used in the compositions and methods. In some embodiments,the eukaryotic transcription/translation system comprises individuallypurified components. Supplementation of the eukaryotictranscription/translation system with spherical endoplasmic reticulumfragments called microsomes enables the production of membrane proteinshaving post-translational modifications such as glycosylation,acetylation, isoprenylation, and phosphorylation.

The formation of a layer of amphipathic molecules around the surfaces ofthe droplets is straightforward. For example, it may be achieved simplyby providing the amphipathic molecules in the hydrophobic medium or inthe aqueous solution of the droplets, whereupon the layer can formnaturally if the droplets are left for a sufficient period of time. Theamphipathic molecules may also be dissolved, or suspended as lipidvesicles in the droplets themselves, from where they again spontaneouslyform monomolecular layers at the interface between the droplet and thehydrophobic medium, that may have an equilibrating concentration of theamphipathic molecule in the hydrophobic medium.

The bilayer is formed simply by bringing droplets into contact with oneanother or by bringing into contact a droplet with a hydrophilicsurface. The orientation of the amphipathic molecules in the layeraround the aqueous solution allows the formation of the bilayer. As thedroplets are brought into contact, after the intervening hydrophobicmedium has been displaced the bilayer forms very quickly as an interfacebetween the contacting droplets. The bilayer forms a roughly planarsurface between the two droplets which are otherwise generallyspherical. This planar bilayer is the shape with the lowest free surfaceenergy and has a negative free energy of formation. Formation of thebilayer at the interface of the two droplets is therefore a spontaneousevent. The amphipathic molecules allow two droplets to be brought intocontact without allowing them to coalesce by the formation of a stablebilayer.

Methods of preparing a droplet-interface bilayer system are disclosed.

The method of forming a system comprising a bilayer of amphiphilicmolecules provided at the interface between two droplets can compriseforming a first droplet of a first aqueous solution in the hydrophobicmedium, the first droplet comprising a layer of amphipathic moleculesaround the surface of the first aqueous solution, and containing atranscription/translation extract and a heterologous polynucleotideencoding a membrane polypeptide; forming a second droplet of a secondaqueous solution in the hydrophobic medium, the second dropletcomprising a layer of amphipathic molecules around the surface of thesecond aqueous solution; bringing the droplets into contact with oneanother in the hydrophobic medium so that a bilayer of the amphipathicmolecules is formed as an interface between the contacting droplets; andincorporating a polymer into the bilayer. In an embodiment, thehydrophobic medium or an aqueous phase contains a polymer that insertsinto the bilayer and incorporating the polymer into the bilayercomprises the polymer self-inserting into the bilayer. In anotherembodiment, incorporating the polymer into the bilayer comprisescontacting the first or the second droplet with a lipid-polymer film ona surface of a substrate in the hydrophobic medium such that alipid-polymer monolayer forms on a surface of the first or seconddroplet. Contacting the first or the second droplet with thelipid-polymer film can occur prior to bringing the droplets into contactwith one another in the hydrophobic medium. The method can furthercomprise coating the substrate with a lipid-polymer solution to form thelipid-polymer film on the surface of the substrate.

Bringing droplets into contact with one another can comprise moving oneof the droplets into contact with the other droplet. The method canfurther comprise incubating the first droplet under conditions such thatthe encoded membrane polypeptide is synthesized and/or incubating thecontacted droplets such that the membrane polypeptide is inserted intothe bilayer. The droplets can be moved while in contact with one anotherto vary the area of the bilayer of the amphipathic molecules. Thedroplets in contact with one another can also be separated.

The droplets may be handled by a variety of techniques. One method ofmoving the droplets disclosed in U.S. Pat. No. 8,268,627 is to disposean anchor having a hydrophilic outer surface inside a droplet. Movementof the anchor allows the droplet to be moved, for example to bring itinto contact with another droplet. One example of an anchor includes anelectrode treated to have a hydrophilic surface to interact with theaqueous droplet.

The formation of the bilayers is highly reversible and repeatable.Droplets which have been brought into contact with one another may befreely separated to divide the bilayer and may be subsequently broughtinto contact again to re-create the bilayer.

The degree of control makes the formation of the bilayers easy tostandardize. In particular, it is easy to vary the area of the bilayerof the amphipathic molecules by moving the droplets when the dropletsare in contact with one another. The change in the area of the bilayersmay be observed visually or by capacitance measurements. The diameter ofthe bilayer has been varied over the range from about 30 μm to about1000 μm, although this is not thought to be the limit.

In addition, the nature of the hydrophobic medium determines the degreeof spreading of the contacted monolayers and thereby the contact angle.For example, for bilayers of glycerylmonooleate (GMO) formed in decaneas the hydrophobic medium, the contact area is relatively small and thecontact angle is about 3° , this being in agreement with contact anglesmeasured in conventional lipid membrane systems. On the other hand, ifthe hydrophobic medium is squalene, a larger contact area is formed andthe contact angle is 25°, again in agreement with measurements onconventional lipid membranes. These solvent-dependent effects reflectthe small free energy of formation of the GMO:decane system (around −4mJ/m²) as compared to the GMO:squalene system (around −500 mJ/m²), wherethe bilayer thickness concomitantly decreased from 50 Å to 25 Å,signifying a depletion of the larger squalene solvent from the bilayer.This non-linear increase in free energy of formation departs from simpleLifshitz theory for two infinite slabs of water acting across the thinoil film, and is more in line with a “depletion flocculation” effect.Essentially, the larger squalene solvent molecules are entropicallyexcluded from the GMO bilayer, and this depletion of solvent exerts agreater osmotic pressure on the bilayer, thereby raising the free energyof formation by orders of magnitude in going from decane to squalene,over and above any Lifshitz effects. Adhesion and the strength andstability of the contact then are largely dependent on the presence orabsence of solvent in the bilayer.

An advantage of the present composition and methods is that the DIBsystem allows the use of a relatively small volume of aqueous solution.In particular, the volume may be smaller than that present in thechambers of a cell used in conventional planar bilayer techniques. Thedroplets may typically have a volume less than about 1000 nL. A dropletdisclosed herein can have a volume of at least about 14 pL and less thanabout 1000 nL, preferably at least about 20 pL and less than about 1000nL, more preferably at least about 100 nL and less than about 800 nL. Ingeneral the droplets may be of any size limited only by the degree ofcontrol of the dispenser of the aqueous solution and the limits ofoptical resolution if direct manipulation is desired. Droplets that arenot required to have electrical recording or stimulus from placedelectrodes can be assembled in suspension forming a raft or 3D aggregateor flocculent of droplets having dimensions of micrometers to evennanometers that are all in contact with each other via their interveningbilayers. Using a standard pipette, droplets having volumes in the rangefrom 200 nL to 800 nL can be prepared. However, droplets of smallervolumes can be produced with suitable equipment. For example, usingmicro-pipette manipulation to form droplets from glass micro-pipettes,observed in a relatively powerful microscope, permits formation ofdroplets of a diameter of about 30 μm and a volume of approximately 14pL. In suspension, droplet aggregation of droplets of diameter of about200 nm yields internal volumes of approximately 4 attoliter (aL, i.e.,10⁻¹⁸ L).

When electrical measurements are to be performed with the droplet,another consideration which can limit desirable droplet size is the needfor an electrical interface. The electrodes require a certain amount ofsurface area in contact with the droplet to permit the flow ofelectricity through the recording equipment. For example, for anelectrode made from a silver wire with a diameter of about 0.1 mm, thedroplets have to be bigger than 0.1 mm in diameter. However use ofsmaller electrodes would allow the droplets to be smaller in diameter.In an embodiment of the compositions and methods disclosed herein, thesize of a droplet would be between 100 and 1000 microns in diameter.Such droplets are easy to manipulate but still inexpensive to use.

The polymer incorporated into the interfacial bilayer of the DIB or themembrane bilayer is an amphipathic polymer that can self-assemble intovesicles in dilute solution. Such self-assembling amphipathic polymerscan self-direct their insertion into lipid bilayers. The amphipathicpolymer can be any amphipathic polymer that does not destabilize theinterfacial bilayer formed between the contacted droplets or inhibitbiological function of the membrane polypeptide inserted into theinterfacial bilayer. The self-assembling amphipathic polymer can be ablock copolymer, preferably a linear block copolymer. A “blockcopolymer” is a copolymer comprising two or more homopolymer subunitslinked by covalent bonds. Each structurally unique homopolymer subunitcan be designated by a capital letter, such as A or B, to designate arepeating unit in the copolymer. The union of the homopolymer subunitsmay require an intermediate non-repeating subunit, known as a junctionblock. Block copolymers with two or three distinct blocks are calleddiblock copolymers and triblock copolymers, respectively. Examples ofsuitable block copolymers for use in the disclosed compositions andmethods include AB diblock or ABA triblock copolymers. The hydrophobicblock can be any hydrophobic polymer that does not result in a blockcopolymer that destabilizes the interfacial bilayer formed between thecontacted droplets or inhibits biological function of the membranepolypeptide inserted into the interfacial bilayer. Examples of suitablehydrophobic block polymers are poly(methyl methacrylate) (PMMA) andsilicones. The silicone polymer can be a polydimethylsiloxane. Thehydrophilic block can be any hydrophilic polymer that does not result ina block copolymer that destabilizes the interfacial bilayer formedbetween the contacted droplets or inhibits biological function of themembrane polypeptide inserted into the interfacial bilayer.

Examples of suitable hydrophilic polymers are poly(ortho ester),polyethylene glycol (PEG), poly(ε-caprolactone-co-lactide) (PCLA), andpoly (2-methyl-2-oxazoline). The polymer can be a tri-block copolymer(ABA) comprising a polydimethylsiloxane (B unit) capped at both endswith a poly(2-methyl-2-oxazoline) (A unit) The length of thepolydimethylsiloxane block can be about a 45-mer to a 110-mer,preferably a 50-mer to a 100-mer, more preferably a 55-mer to a 90-mer,yet more preferably a 58-mer to an 80-mer, even more preferably a 60-merto an 70-mer, and most preferably a 65-mer. The length of a poly(2-methyl-2-oxazoline) capping unit can be about a 2-mer to a 15-mer,preferably a 3-mer to a 12-mer, more preferably a 4-mer to a 10-mer, yetmore preferably a 5-mer to an 8-mer, and most preferably a 6-mer. Theamphipathic polymer can be purchased commercially or synthesized.Further examples of suitable amphiphilic polymers are disclosed forexample in WO2014/064444 and U.S. Pat. No. 6,723,814, herebyincorporated by reference.

The amphipathic polymer can be present in either or both droplets or inthe hydrophobic medium. The amphipathic polymer can be present in thedroplets or the hydrophobic medium in an amount effective forstabilization of the bilayer of the IVTT-containing DIB. The phrase“amount effective for stabilization” means an amount promotingstabilization of the IVTT-containing DIB such that the DIB is stable forat least 30 minutes without the two droplets coalescing, but does nothinder membrane protein incorporation into the interfacial bilayer ofthe DIB. For example, when present in the hydrophobic medium, theamphipathic polymer can be at a concentration greater than about 0.01g/L, greater than about 0.02 g/L, greater than about 0.05 g/L, at leastabout 0.07 g/L, at least about 0.09 g/L, at least about 0.1 g/L, atleast about 0.15 g/mL, at least about 0.2 g/mL or at least about 0.25g/mL.

The amphipathic polymer can be present in a lipid-polymer film on asurface of a substrate. The amphipathic polymer can be present in thelipid-polymer solution used to make the film at a concentration at leastabout 0.15 g/mL, at least about 0.2 g/mL or at least about 0.25 g/mL, atleast about 0.5 mg/mL.

The lipid-polymer film can be formed on a surface of the substrate bycoating the substrate with a lipid-polymer solution. The lipid-polymersolution can be applied by any suitable method. Such methods includepipetting the solution onto the surface followed by evaporation ofsolvent, spin coating, spray deposition, or stamping. The substrate canbe any suitable material that does not dissolve in the solvent or absorbthe lipid or polymer, e.g., glass, silicon, or other smooth chemicallyinert surface, preferably glass, having at least one planar surface.

Alternatively a porous substrate can be used to form the lipid-polymermonolayer on a surface of a droplet. Examples of a porous substrateinclude alumina and a polymer membrane. Without being bound by theory,it is believed that a porous substrate would serve as a sponge to soakup the lipid-polymer for future deposition to droplets.

In contacting a droplet with the lipid-polymer film on a surface of asubstrate in the hydrophobic medium, the droplet can be attached to asupport, e.g., an electrode, in the hydrophobic medium. The droplet canbe rolled over the film on the substrate surface to transfer a monolayerof lipids and polymer from the film to at least a portion of themonolayer enveloping the droplet. The substrate is submerged in thehydrophobic medium such that the film is presented for contact with adroplet. The film can be presented for contact by placement of thesurface at an angle of 25 to 65°, preferably 30 to 60°, more preferably35 to 55°, yet more preferably 30-50 °, most preferably about 45°relative to the surface of the hydrophobic medium. Contacting thedroplet with the lipid-polymer film can occur prior to or after bringingthe two droplets into contact with one another in the hydrophobicmedium.

Lipids in the lipid-polymer solution can comprise any lipids disclosedherein. In an embodiment, the lipid in the lipid-polymer solution caninclude 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), or a mixturethereof. In an embodiment, the lipid-polymer solution comprises a massratio of 1,2-DPhPC:DPhPE:polymer of 20:5:0.5 to 20:5:10, preferably20:5:1 to 20:5:5, most preferably 20:5:1.

Another advantage of the present compositions and methods is that it ispossible to bring more than two droplets into contact with each other ina chain or network, for example on a flat or dimpled surface, in amicrofluidic channel or, in aggregated or flocculated suspension. Thesimplicity and control with which the bilayers can be formed simply bymoving droplets around makes it straightforward to build large chains ornetworks which would be impractical in a system where bilayers areformed in apertures in barriers in accordance with conventionaltechniques. This opens up the possibility of studying much largersystems than is practical with the conventional technique, for examplemodelling entire systems using multiple droplets.

The droplets and bilayer can be made with a wide range of materials.

In general, the amphipathic molecules can be of any type which form abilayer in the hydrophobic medium in which the droplets are positioned.This is dependent on the nature of the hydrophobic medium and theaqueous solution, but a wide range of amphipathic molecules arepossible. “Amphipathic” molecules are molecules which have bothhydrophobic and hydrophilic groups. Herein, “amphipathic” and“amphiphilic” are used synonymously. The layer formed around the dropletis a monolayer of amphipathic molecules which is formed and maintainednaturally by the interaction of the hydrophobic and hydrophilic groupswith the aqueous solution so that the molecules align on the surface ofthe droplet with the hydrophilic groups facing inwards and thehydrophobic groups facing outwards.

Examples of amphipathic molecules include hydrocarbon based surfactantsand a variety of biological compounds such as phospholipids,cholesterol, glycolipids, fatty acids, bile acids, and saponins.Examples of hydrocarbon based surfactants, include sodium dodecylsulfate (anionic), benzalkonium chloride (cationic), cocamidopropylbetaine (zwitterionic), and 1-octanol

One important class of amphipathic molecules which may be used informing droplets is lipid molecules. The lipid molecules may be any ofthe major classes of lipid, including fatty acids, glycerolipids,glycerophospholipids, sphingolipids, sterol lipids, prenol lipids,saccharolipids and polyketides. Some important examples include aphospholipid, a glycolipid and cholesterol. The lipid molecules may benaturally occurring or synthetic.

Lipids forming the lipid bilayer or in the lipid-polymer solution cancomprise any suitable lipids. Example lipids include1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE). The lipidstypically comprise a head group, an interfacial moiety and twohydrophobic tail groups which may be the same or different. Suitablehead groups include, but are not limited to, neutral head groups, suchas diacylglycerides (DG) and ceramides (CM); zwitterionic head groups,such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) andsphingomyelin (SM); negatively charged head groups, such asphosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol(PI), phosphatic acid (PA) and cardiolipin (CA); and positively chargedheadgroups, such as trimethylammonium-Propane (TAP). Suitableinterfacial moieties include, but are not limited to,naturally-occurring interfacial moieties, such as glycerol-based orceramide-based moieties.

The amphipathic molecules need not be all of the same type. Theamphipathic molecules may be mixtures. The monolayer around a givendroplet can comprise a single amphipathic molecule or a mixture ofamphipathic molecules. Furthermore, the amphipathic molecule or mixtureof amphipathic molecules in the respective outer layers of the twodroplets brought into contact can be the same or different. For example,the amphipathic molecules in the respective outer layers of the twodroplets brought into contact can be of different types or two differentmixtures so that the bilayer formed by the two monolayers is asymmetric.

The aqueous solution of each droplet may be freely chosen as appropriatefor the experimental study which is to be performed. The first aqueoussolution and the second aqueous solution of the two droplets may be thesame or different. The nature and concentration of the solutes can befreely varied to vary the properties of the solution. One importantproperty is pH and this can be varied over a wide range. Anotherimportant point in experiments using electrical measurements is toselect appropriate salts to carry the current. Another importantproperty is osmolarity.

Herein, a “hydrophobic medium” means a water-immiscible solvent. Thehydrophobic medium can also be selected from a wide range of materials.The material is hydrophobic so that the aqueous solution forms a dropletrather than mixing with the hydrophobic medium, but otherwise thehydrophobic medium can be freely chosen. The viscosity of thehydrophobic medium can be selected to affect the movement of thedroplets and the speed of formation of the layer of amphipathicmolecules around the aqueous droplet in the case that they are providedin the hydrophobic medium.

The hydrophobic medium may be an oil. Any type of oil is suitable aslong as its surface activity is relatively high, and it does notdestabilize the formed bilayer of the contacted droplets. The oil may bea hydrocarbon which may be branched or unbranched, for example ahydrocarbon having from 5 to 20 carbon atoms (although hydrocarbons oflower molecular weight would require control of evaporation). Suitableexamples include alkanes or alkenes, such as hexadecane, decane,pentane, or squalene. Other types of oil are also possible. For examplethe oil may be a silicone or a fluorocarbon. A hydrophobic mediumcomprising a silicone oil or a fluorocarbon oil might be useful for thestudy of some systems, for example to minimize loss of a particularmembrane protein or analyte from the droplet or to control gas contentsuch as oxygen. The oil can be a single compound or a mixture ofcompounds.

A membrane polypeptide can be synthesized within the droplet containingthe IVTT or provided in one or more of the droplets for insertion intothe bilayer. The present method does not limit the choice of membranepolypeptide, provided that the aqueous solution is chosen withappropriate properties for the protein in question. Thus the membranepolypeptide may be of any type. The membrane polypeptide can be aprokaryotic or eukaryotic polypeptide. The membrane polypeptide can bean integral membrane protein or a peripheral membrane protein. Thedisclosed methods and compositions apply to any membrane proteinsincluding the two major classes that are β-barrels or α-helical bundles.An important application is a membrane polypeptide which is a pore or achannel. Examples of membrane polypeptides include a polypeptide pore orchannel, a receptor, a transporter, or a protein which effects cellrecognition or a cell-to-cell interaction. In preferred embodiments, themembrane polypeptide is a pore or channel. Suitable pores that may beemployed in the system or composition include for example MspA, α-HL,CsgG, lysenin, and homologues and paralogues thereof, such as disclosedin WO2010/0034018 and WO2016/034591. However other pores well known inart may be employed.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a molecule formed from the linking,in a defined order, of at least two amino acids. The link between oneamino acid residue and the next is an amide bond and is sometimesreferred to as a peptide bond. A polypeptide can be obtained by asuitable method known in the art, including isolation from naturalsources, expression in a recombinant expression system, chemicalsynthesis, or enzymatic synthesis. The terms also apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymers.

Unless otherwise indicated, a particular polypeptide sequence alsoimplicitly encompasses conservatively modified variants thereof. Aconservative amino acid substitution in a polypeptide sequence includesthe substitution of an amino acid in one class by an amino acid of thesame class, where a class is defined by common physicochemical aminoacid side chain properties and high substitution frequencies inhomologous proteins found in nature, as determined, for example, by astandard Dayhoff frequency exchange matrix or BLO SUM matrix. Sixgeneral classes of amino acid side chains have been categorized andinclude: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III(Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val,Met); and Class VI (Phe, Tyr, Trp). For example, substitution of an Aspfor another class III residue such as Asn, Gln, or Glu, is aconservative substitution. One of skill in the art can readily determineregions of the molecule of interest that can tolerate change byreference to Hopp/Woods and Kyte-Doolittle plots.

The term “nucleic acid” or “polynucleotide” includes DNA molecules andRNA molecules. A polynucleotide may be single-stranded ordouble-stranded. Polynucleotides can contain known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs). A polynucleotide can be obtaine by asuitable method known in the art, including isolation from naturalsources, chemical synthesis, or enzymatic synthesis. Nucleotides may bereferred to by their commonly accepted single-letter codes. In preferredembodiments, the polynucleotide encodes a polypeptide, and can beenzymatically transcribed and/or translated.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The IVTT-containing droplet-interface bilayer system can be used toperform experiments involving a process occurring at or through thebilayer of the amphipathic molecules. A major class of experiments usesa membrane protein inserted into the bilayer. This may be achievedsimply by providing the membrane polypeptide in the first or secondaqueous solution. The membrane polypeptide can be provided by synthesiswithin the first droplet by the IVTT system therein. After the formationof the bilayer, the membrane polypeptide naturally inserts into thebilayer in the same manner as with a bilayer formed by conventionaltechniques.

It has been observed that the bilayer behaves functionally in the samemanner as a bilayer formed by conventional techniques. Therefore thebilayer formed by the present methods can be used to perform the sametypes of experiments, but providing a number of advantages which broadenthe range of possible experiments, as discussed further below. Thus thepresent method may be applied to a wide range of experiments includinginvestigation and/or screening of membrane proteins, investigationand/or screening of analytes which interact with membrane proteins, andinvestigation and/or screening of the bilayers. Indeed the method may beused to study any bilayer phenomena in general, typically involving aprocess occurring at or through the bilayer.

Thus, methods of analyzing membrane polypeptide function are disclosed.

The method of analyzing membrane polypeptide function comprisescontacting a test compound with the DIB system disclosed herein; andmeasuring a detectable signal from the system in the presence and in theabsence of the test compound. The method can further comprise bringingelectrodes into electrical contact with the droplets when the dropletsare in contact with one another and measuring an electrical signal usingthe electrodes.

Measuring a detectable signal from the system can mean measuring anelectrical property, measuring a change in an ion concentration,measuring a change in protein conformation, measuring binding of a testcompound to the membrane protein, measuring a change in phosphorylationlevel, measuring a change in second messenger level, measuring a changein neurotransmitter level, measuring a change in a spectroscopiccharacteristic, measuring a change in a hydrodynamic (e.g., shape)property, measuring a change in a chromatographic property, or measuringa change in solubility. In preferred embodiments, the detectable signalis an electrical signal or a signal from a chromophore.

The test compound can be a small molecule or a biological moiety, suchas a protein, a sugar, a nucleic acid, or a lipid.

The following examples are merely illustrative of the compositions andmethods disclosed herein and are not intended to limit the scope hereof.

EXAMPLES Example 1 Preparation of a Stable DIB System Containing aEukaryotic IVTT Expressing a Membrane Protein in situ

A eukaryotic IVTT containing DIB system is prepared for expressing ahuman ion channel in situ. A stabilizing polymer is included in theinterfacial bilayer of the DIB.

A THERMO SCIENTIFIC 1-Step Human Coupled IVT Kit—DNA (Catalog No. 88882)was used as the IVTT system in the experiments below. This THERMOSCIENTIFIC kit is a mammalian in vitro translation (IVT) system based onHeLa cell lysates. The kit contains all of the cellular componentsrequired for protein synthesis, including ribosomes, initiation factors,elongation factors and tRNA. When supplemented with the proprietaryaccessory proteins and reaction mix included in the kit and with a DNAtemplate, this IVTT system can synthesize protein from the DNA template.

First, two Ag/AgCl electrodes were prepared from 2 cm lengths of 100 μmdiameter silver wire. Each wire was briefly melted over a flame tocreate an approximately 250 μm diameter ball at the end. These wereimmersed in a solution of sodium hypochlorite until the silver turneddark grey, indicating a layer of AgCl had covered the electrode surface.The ball ends of the electrodes were then coated with a layer oflow-melt agarose in buffer; this rendered the electrode surfaceshydrophilic and capable of holding aqueous droplets. Each electrode wasfixed by an alligator clip, which in turn was attached to an NMN-213-axis micromanipulator (Narishige).

The electrodes were submerged in a homemade acrylic plastic cup-shapedchip filled with hexadecane containing 0.1 mg/mL of 6-65-6polydimethylsiloxane (PDMS) polymer. IVTT and lipid vesicles were mixedin varying proportions and then 200 nL droplets were manually pipettedonto the grounded (cis) electrode in most cases. Lipid vesicles wereusually placed on the opposing (trans) electrode by manually pipettingdroplets onto the electrode. The presence of fully formed lipidmonolayers was confirmed visually using a stereomicroscope: the dropletsdroop from the electrodes following monolayer acquisition. After fixingthe IVTT droplet to the cis electrode, the electrode was guided viamicromanipulator through the chip to form a DIB with the lipid vesicledroplet. The formation of the DIB was monitored by a capacitancemeasurement and generally occurred within less than a minute aftercontacting the droplets. The DIB normally increased in size to greaterthan 300 pF; the droplets were then moved apart slightly to adjust thebilayer size to 300 pF. If a DIB did not form after 5-10 minutes, anapplied potential of >100 mV was used to stimulate bilayer formation.

The THERMO SCIENTIFIC kit reagents are thawed on ice 15 minutes beforethe experiment starts. Meanwhile the coated electrodes are balanced bythe vesicle-only solution. The IVTT solution is prepared throughhierarchical addition of 5 μL HeLa cell lysate, 1 μL accessory proteins,0.8 μL hERG channel-expressing plasmid (Genscript) and 2 μL reactionmix, respectively, to a nuclease-free microtube. After about twominutes, 3.8 μL solution of 4 mM lipid vesicles (at a 4:1 ratio of1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) to1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE) in B1 buffer(120 mM KCl, 60 mM NaCl, 10 mM MgCl₂ and 10 mM HEPES) is added to theIVTT solution. The solution in the microtube is gently mixed by hand toavoid formation of air bubbles. The final solution incubates for threeminutes at room temperature. Then 0.20 μL of the prepared IVTT solutionis injected onto the trans-electrode and a 0.20-μL vesicle-only solutionof 2 mM lipid vesicle (4:1 DPhPC to DPhPE ratio) in B1 buffer isinjected onto the cis-electrode. Taking into account the requiredrelaxation time, on average the droplets are hanging between 3-4 minutesin the oil phase, followed by gentle touching of the two aqueousdroplets using mechanical micromanipulators as previously described inHolden M. A. et al. (J. Am. Chem. Soc. 2007, 129(27): 8650-8655). Thevolume percent of the IVTT droplet is composed of 70% IVTT solution and30% vesicle solution of 4 mM lipid concentration (with 4:1 DPhPC toDPhPE ratio) containing B1 buffer.

After 8-10 minutes of in situ expression of the channel Kv11.1, (encodedby hERG gene) in the DIB, electrophysiological analysis of channelactivity is begun. The electrodes are connected to a patch-clampamplifier (Axopatch 200B; Molecular Devices LLC Axon Instruments) formeasurement of ion channel currents, which are digitized with a Digidata1442A (Molecular Devices LLC) at a sampling rate of 20 kHz. The datafrom the original recording are digitally lowpass filtered at 200 Hz.The oil reservoir and amplifying headstage are enclosed in a metal box,which serve as a Faraday cage to reduce noise. After the experiment, thedroplets can be separated using the micromanipulators.

The results of such an experiment are shown in FIG. 4. The trace showsthe activity of a single channel of Kv11.1 at −50 mV in B1 buffer. Afterchannel insertion into the bilayer, channel activity could be observedfor more than 40 minutes. The single channel conductance was similar tothat observed by others (Portonovo, S. A. et al. Biomed. Microdevices2013, 15 (2), 255-259) and switched between open and closed states (aprocess known as gating) as expected (Helliwell, R. M. “Recording hERGPotassium Currents and Assessing the Effects of Compounds Using theWhole-Cell Patch-Clamp Technique.” Potassium Channels. Humana Press,2009. 279-295.' Vijayvergiya, V. et al. Biomed. Microdevices 2015, 17(1), 12).

Example 2 Preparation of a DIB System Containing a Eukaryotic IVTT and aMembrane Protein in the Presence and Absence of a Stabilizing Polymer

Experiments were performed to determine the stability of a DIBcontaining a eukaryotic cell extract and a stabilizing polymer. In theseexperiments an IVTT-containing DIB is prepared that includes a poreprotein, the toxin α-hemolysin (α-HL), in one droplet for testing thestability of the DIB system in the presence and absence of a stabilizingpolymer.

A droplet containing the IVTT was formed by first mixing 70% v/v of anIVTT solution containing 5 μL HeLa cell lysate, 1 μL accessory proteins,0.8 μL GFP-DNA, and 2 μL reaction mix from a THERMOSCIENTIFIC 1-StepHuman Coupled IVT Kit—DNA with 30% v/v of a γ-Cyclodextrin (γCD;Sigma-Aldrich Cat no. C4930)-containing lipid vesicle solution (with a4:1 DPhPC to DPhPE ratio in B1 buffer). The final γCD concentration inthe IVTT droplet is 50 μM and the final lipid vesicle concentration(with 4:1 DPhPC to DPhPE ratio) is 0.8 mM. A second droplet containing0.2 μg/mL α-hemolysin) and lipid vesicles made from 2 mM of 4:1 DPhPC toDPhPE ratio in buffer (120 mM KC1, 60 mM NaCl, 10 mM MgCl₂ and 10 mMHEPES) was then injected to an oil bath containing 0.1 mg/mL ofstabilizing polymer. The heptameric hemolysin was provided as a kindgift from Oxford Nanopore Technologies. The stabilizing polymer in thisexperiment was a triblock copolymer, a 65 mer of polydimethylsiloxanecapped at both ends with a 6 mer of poly(2-methyl-2-oxazoline) (“TBCP”;gift from Oxford Nanopore Technologies).

Cyclodextrin acts as a reversible blocker of the α-hemolysin pores. Inthe experiment, the characteristic binding behavior of cyclodextrin tothe α-hemolysin helps to distinguish between conductance through themembrane-inserted toxin α-hemolysin and other pores which might form dueto membrane damage.

The electrophysiological recording obtained from observing a singleα-hemolysin pore for 80 minutes in this IVTT-containing DIB system isshown in FIG. 2. The DIB was stable throughout this time except forbrief periods of high conductance (marked by black triangles in FIG. 2).After each spike, the DIB spontaneously returned to a stable baselineand subsequent pore activity was not affected. The inset of FIG. 2 showsa small expanded section of the recording (black bar) to show thea-hemolysin pore blockades by cyclodextrin.

FIGS. 3A, 3B, 3C, 3D present graphs illustrating expanded regions ofFIG. 2 marked with black triangles showing current spikes observed inpolymer-stabilized IVTT-DIBs. Some spikes are larger than others and allappear to “self-heal” within 5 seconds. Pore blockades by cyclodextrinare observed both before and after each spike, suggesting that thespikes do not affect pore activity.

The toxin a-hemolysin is only able to form conducting ion pores inmembranes. If a film thicker than a lipid bilayer was formed in thepresence of the amphipathic polymer, no pore activity would be seen.However, in all experiments α-hemolysin activity is observed in thepolymer-stabilized DIBs in the presence of the HeLa lysate. For example,a single pore was introduced into a polymer-stabilized IVTT-DIB and itsactivity was monitored for more than 80 minute (FIG. 2). The magnitudeand lifetime of the reversible cyclodextrin blockades of the pore wereconsistent with previous reports. Therefore, the presence of the polymerdoes not affect the cyclodextrin-pore interaction.

We noted several interesting features of polymer-stabilized IVTT-DIBs.First, the capacitance of the DIB was far lower in the presence of thepolymer. The capacitance of a membrane is directly proportional to itsarea while inversely proportional to its thickness. As we were routinelyable to insert pores into the triblock polymer stabilized DIBs, thethickness of the membrane could only be marginally affected. Weattribute the low capacitance to relatively small DIB area.

Second, in most recordings we observed an abrupt end to the poreactivity after at least an hour. In the example of FIG. 2, theelectrical activity dropped to the baseline current at approximately 80minutes (FIG. 2, end of recording). One possible explanation is that thepolymer may continue to partition into the DIB over time, thickening themembrane to the point where pores cannot be observed. In addition, thesurface properties of the droplets change over time, developing anoticeable “shell” that is not seen in the absence of polymer. Creasesand wrinkles were seen in the droplets' surface when they werecompressed by a pipette tip. These observations suggest that thepolymer-droplet interaction evolves over the course of the experiment,changing the properties of the DIB. These changes are unlikely to hinderion channel-drug interaction studies, since the test would be completedbefore the membrane would no longer be usable. Specifically, we expectprotein synthesis to be completed within 20 minutes, with channelrecording immediately following for at most 10 minutes. We wereregularly able to record for more than 1 hour of pore activity.

The most striking feature of the polymer-stabilized IVTT-DIB is itsability to self-heal defects. Several large spikes in current wereobserved during our experiments with the polymer stabilized IVTT-DIBs.Typical examples are highlighted with black triangles in FIG. 2. Uponcloser inspection, one can see a rapid rise in current followed by arapid decrease back to the single-pore current level (FIG. 3). Note thatpore blockades by cyclodextrin are observed both before and after eachspike, which suggests that the spike does not affect protein activity.The current spikes do not occur in the absence of the IVTT mixture.

In the absence of the polymer, we were not able to form stableIVTT-DIBs. In experiments in the absence of the polymer, one dropletcontained 70% v/v IVTT solution (HeLa lysate, accessory proteins,reaction mixture and GFP-DNA) and 30% v/v aqueous solution of lipidvesicle (4 mM) with γ-cyclodextrin (50 μM) dissolved in B1 buffer whilethe other droplet contained lipid vesicles (1.3 mM) and α-HL (0.2 m/mL)in B1 buffer. The hydrophobic phase is hexadecane as above, but withoutthe TBCP. IVTT-DIBs formed, but pore activity could be recorded for onlybrief periods of time in the absence of the polymer. The longestrecording we obtained in the absence of the stabilizing polymer was only100 s. The recording from that experiment is shown in FIG. 5, whichshows the recording of the current from the time of initial contact ofthe two droplets (time 0 s) to the time at which destabilization andcoalescence of the drops occurs. Signals were filtered with a 200 Hzlowpass Bessel filter.

Immediately after formation of the DIB, pores insert into the bilayer.As can be seen in FIG. 5, reversible blockages of the pores bycyclodextrin are observed, starting at about 60 sec after dropletcontact, but the recording shows that DIB destabilization andcoalescence occurs less than a minute later. The trace in FIG. 5abruptly ends and the current shoots off scale because the dropletscoalesce. In comparison to the DIBs including the stabilizing polymer,there are no signs of self-healing in the recording.

In another control experiment, the IVTT-containing DIB was prepared asabove, in the absence of the stabilizing polymer, however in thisexperiment the two aqueous droplets were injected on the electrodesright after taking the aliquots from ice. Due to the lower initialtemperature of the two droplets, it takes a few minutes for the toxin tobe incorporated into the bilayer. The electrophysiology recording forthis experiment (FIG. 6) shows that before the toxin is incorporatedinto the bilayer, the bilayer is unstable and the two aqueous dropletscoalescence without any sign of current leakage.

Similar membrane instability has been observed using other eukaryoticIVTT systems, including rabbit reticulocyte lysate, wheat germ extract,and yeast extract.

Inclusion of a stabilizing polymer in the bilayer appears to prevent thegrowth or expansion of membrane defects and may even “plug” defects asthey form.

In these experiments to test for DIB stability, the pore protein α-HLwas not synthesized in situ. However, a plasmid to express greenfluorescent protein (GFP) was included in the IVTT mixture beforeforming these DIBs to act as a control for IVTT function under theexperimental conditions. After each experiment, the fluorescence of theIVTT droplet was measured. In all cases, GFP was expressed. Importantly,neither the presence of the stabilizing amphipathic polymer nor theaddition of vesicles and buffering salts affected the IVTT expression ofGFP, demonstrating that the conditions required for DIB stability arecompatible with the IVTT expression of eukaryotic proteins.

The data suggest that that polymer/lipid interaction may be dynamic,able to self-heal and change over time. Ideally, the polymer wouldconstitute only a small fraction (less than 10%) of the DIB, since thelipids represent the most biomimetic environment for channel analysis.

In these experiments, concentrations of less than or equal to 0.05 g/LTBCP in the hexadecane bath did not stabilize the IVTT-DIB. Theelectrophysiology recording of an experiment including 0.05 g/L TBCP inthe hexadecane bath is shown in FIG. 7. All other conditions areidentical to those for the experiment of FIG. 2. As can be seen in FIG.7, two distinct attempts of self-healing happen. Apparently, this amountof polymer is not enough to hold the bilayer together. After the firstself-healing attempt, the toxin gets incorporated into DIB (the one-stepcurrent increase), however in the absence of enough TBCP molecules theintegrity of DIB is compromised.

Example 3 Addition of Polymer by Rolling Method

In order to fix the polymer/lipid ratio at the start of a DIBexperiment, we developed a method for adding polymer to the bilayers inwhich no bulk polymer is dissolved in the hexadecane phase. Whenincluding the polymer in the oil bath, the polymer can continue topartition into the DIB during the experiment and will eventuallyseparate the two leaflets. Thus, the membrane changes over time. Withoutbeing bound by theory, it is believed that because the rolling methoddoes not include excess polymer in the oil, the initial DIB monolayersremain at a constant polymer/lipid ratio throughout the experiment.

Reagents. The 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) lipidand 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE) lipid wereobtained from Avanti Polar Lipids (Alabaster, AL). Hexadecane and lowmelting point agarose were obtained from Sigma-Aldrich. Theγ-cyclodextrin, silicone oil, sodium chloride, potassium chloride,magnesium chloride and HEPES buffer were obtained from FisherScientific. The 1-Step Human Coupled IVT Kit—DNA (Kit Contains HeLalysate, accessory proteins, reaction mix, positive control DNA: pCFE-GFPand pT7CFE1-CHis) was purchased from ThermoFisher Scientific. Wild Typeα-hemolysin pre-heptamer and triblock copolymer (TBCP) were kind giftsfrom Oxford Nanopore Technologies.

Preparation of vesicles, toxin/blocker solutions and IVTT solution. Allvesicles solutions were prepared in B1 buffer (10 mM HEPES, 120 mM KCl,10 mM MgCl2 and 60 mM NaCl buffered to pH 7.2 with sodium hydroxide). A4 mM DPhPC:DPhPE (4:1) vesicle solution was prepared by drying analiquot of DPhPC (in pentane) mixed with DPhPE (in chloroform) driedunder a stream of nitrogen until the solvent evaporated, followed byfurther drying in a vacuum desiccator for 2 hours. Lipids werere-suspended in B1 buffer and extruded once through a polycarbonatefilter with 100 nm track-etched pores (Millipore) using a mini-extruder(Avanti). This solution was used to prepare the IVTT-containing droplet.

Final α-hemolysin concentration in the droplet was achieved throughserial dilution of a stock solution (20 μg/mL) to 2 μg/mL with 18 MΩwater, followed by a final dilution to the desired concentration to beused in the droplet with B1 buffer. γ-cyclodextrin solution, used asblocker, also was diluted from a stock solution (10 mM) through serialdilution to a final concentration of 500 μM with 18 MΩ water. The lastdilution was made with vesicle solution (2 mM, DPhPC: DPhPE) to achievea final concentration of 50 μM.

Upon receiving the 1-Step Human Coupled IVT Kit, reagents were aliquoted(EPPENDORF LOBIND microcentrifuge tubes) and immediately frozen inliquid nitrogen and stored at −80° C. Each aliquot was prepared to havereagents sufficient for two reactions. The IVTT solution was preparedfrom the 1-Step Human Coupled IVT Kit—DNA by mixing 5 μL HeLa lysate, 1μL accessory proteins, 2 μL reaction mix and 0.8 μL pCFE-GFP DNAtogether after thawing the aliquots of each solution in ice. Thissolution was used to prepare IVTT-containing droplets containing 70% volIVTT mixture and 30% vesicle solution (or just B1 buffer in a rollingexperiment).

Droplet-interface bilayer formation. Two Ag/AgCl electrodes wereprepared from 2 cm lengths of 100 μm diameter silver wire. Each wire wasbriefly melted over a flame to create an approximately 250 μm diameterball at the end. These were then immersed in a solution of sodiumhypochlorite until the silver turned dark grey, indicating a layer ofAgCl had covered the electrode surface. The ball ends of the electrodeswere then coated with a layer of low-melt agarose in buffer (2 wt %);this rendered the electrode surfaces hydrophilic and capable of holdingaqueous droplets. Each electrode was fixed by a clip, which in turn wasattached to an NMN-21 3-axis micromanipulator (Narishige).

Polymer-stabilized DIB; Polymer in oil bath method. The electrodes aresubmerged in an oil pool (1.2 mL) contained in a homemade acrylicplastic chamber. The composition of the oil is 96% vol hexadecane and 4%vol silicone oil with a final polymer concentration of 0.1 mg/mL. 200 nLdroplets of either IVTT-containing complex solution or vesicles aremanually pipetted onto the electrodes. Here, the IVTT-containing dropletis composed of 70% vol IVTT mixture (as described above) and 30% volvesicle solution. The other droplet contained only vesicle solution. Inall trials, to examine toxin activity through reversible γ-cyclodextrinblock, α-Hemolysin is added to droplets (cis or trans) as part of avesicle solution with a final concentration of 0.1 μg/mL. Similarly,γ-cyclodextrin is added to droplets (trans or cis) with a finalconcentration of 50 μM. The electrophysiology conditions are describedbelow.

Polymer-stabilized DIB; Rolling approach. To restrict the amount ofvesicles inside the droplet as a way of inhibiting the unwantedlipid-protein interactions or probable protein sequestering inside lipidvesicles, both cis and trans droplets were prepared without the additionof vesicle solution. B1 buffer was used to prepare both theIVTT-containing droplet (30% vol B1) and the counterpart droplet. Inthis case, the oil bath was 100% vol hexadecane without the addition ofTBCP. The monolayer formation was achieved through rolling of thedroplets over a glass coverslip (Fisher Scientific-φ=10 mm) coated witha lipid-polymer film. DPhPC in pentane, DPhPE in chloroform and TBCP inchloroform was mixed together to reach a final mass ratio of 20:5:1respectively (200 μl DPhPC (10 mg/mL)+50 μl DPhPE (10 mg/mL)+100 μl TBCP(1 mg/mL)). 30 μl of the described solution was carefully placed on thecover slip and without any agitation solvents were evaporated by anitrogen stream. To remove trace amounts of solvent, the coat wasvacuum-dried for 5 hours. After submerging the electrodes (withdroplets) into the hexadecane bath, droplets were placed in one side ofthe chamber and with the aid of a curved tweezer, the cover slip wassubmerged at about a 45° angle (FIG. 11, panel (a)). In less than 2minutes both droplets were rolled side-to-side and also up and down topermit monolayer acquisition. After removing the glass slide, dropletswere left to relax. With this approach IVTT-containing droplets relaxedmuch faster (˜30 seconds) in comparison to buffer-only droplet.

Electrophysiology. The electrodes were connected to a patch-clampamplifier (Axopatch 200B; Axon Instruments). The currents were filteredwith a low-pass Bessel filter (200 dB/decade) with a corner frequency of2 kHz and then digitized with a DigiData 1400 series A/D converter (AxonInstruments) at a sampling frequency of 5 kHz. The oil reservoir andamplifying head-stage were enclosed in a metal box, which served as aFaraday cage. In both experimental approaches, the electrodes wereguided via micromanipulator to form a DIB with the two droplets.Furthermore, the presence of fully formed lipid monolayers was confirmedvisually using a stereomicroscope: the droplets droop (i.e. becomerelaxed) from the electrodes following monolayer acquisition. Theformation of the DIB was monitored by a capacitance measurement andgenerally occurred within less than a minute after contacting thedroplets. For all trials a potential of −70 mV was used unless otherwisementioned. By convention, the IVTT droplet was always placed on thegrounded electrode (cis) and the vesicle droplet was placed on theworking electrode (trans). On occasion, a DIB membrane would ruptureduring experiment, causing the current to jump instantly to an off-scalevalue. When this occurred, the contaminated electrodes were discardedand replaced with fresh Ag/AgCl electrodes. However, the oil bath waskept and the fused solution was dropped to the bottom of chamber throughgentle removal of the electrode from the oil bath. No data was obtainedfrom a ruptured DIB.

A polymer/lipid mixture of ratio 20:5:1 (DPhPC:DPhPE:TBCP) in chloroformwas deposited on a glass surface, as described above, and dried. Thiswas submerged in hexadecane and the droplets were rolled on its surfaceto pick up the material for monolayer formation. After several rolls,the droplets were suspended from the electrodes and brought intocontact. Using this approach, stable DIBs containing IVTT were createdand α-hemolysin activity was monitored as before (FIG. 8).Concentrations of hemolysin (up to 10 mg/mL) that would rupture a DIBwith polymer incorporated by the method in which the polymer is presentin the bulk oil bath (FIG. 9).

Having noted the wrinkles on the droplets and the extraordinarytolerance of polymer-stabilized DIBs made by the droplet-rolling methodto IVTT and high toxin concentrations, we were curious to see whetherthese DIBs could withstand direct mechanical perturbations. Normally, alipid-only DIB will coalesce if a volume is pumped into it. Thus,exchanging the contents of a DIB is a delicate operation. However, a DIBstabilized by TBCP using the droplet-rolling method withstood theinjection and withdrawal of 0.5 μL by pipette (FIG. 10). Indeed, thisprocess was repeated for three cycles without breaking the DIB asverified by subsequent electrical recording.

The compositions and methods disclosed herein include at least thefollowing embodiments:

Embodiment 1. A pair of droplets in a hydrophobic medium comprising afirst droplet of a first aqueous solution in the hydrophobic medium, thefirst droplet comprising a layer of amphipathic molecules around thesurface of the first aqueous solution, and containing atranscription/translation extract and a heterologous polynucleotideencoding a membrane polypeptide; and a second droplet of a secondaqueous solution in the hydrophobic medium, the second dropletcomprising a layer of amphipathic molecules around the surface of thesecond aqueous solution; the first droplet and the second droplet beingin contact with one another such that a bilayer of the amphipathicmolecules is formed as an interface therebetween; an amphipathic polymerand optionally the encoded membrane polypeptide are inserted into thebilayer.

Embodiment 2: The pair of droplets of embodiment 1, wherein the membranepolypeptide is a channel or a pore.

Embodiment 3. The pair of droplets of embodiment 1 or 2, wherein thehydrophobic medium is oil.

Embodiment 4. The pair of droplets of embodiment 3, wherein the oil is ahydrocarbon oil, a silicone oil, a fluorocarbon oil, or a combinationthereof.

Embodiment 5. The pair of droplets of any one of embodiments 1 to 4,wherein the amphipathic molecules are lipid molecules.

Embodiment 6. The pair of droplets of any one of embodiments 1 to 5,wherein each droplet has a volume of at least about 20 6pL and less thanabout 1000 nL.

Embodiment 7. The pair of droplets of any one of embodiments 1 to 6,wherein the first or second droplet has a volume greater than or equalto 100 nL.

Embodiment 8. The pair of droplets of any one of embodiments 1 to 7,wherein the first or second droplet has a volume less than or equal to800 nL.

Embodiment 9. The pair of droplets of any one of embodiments 1 to 8,wherein the polymer is a silicone.

Embodiment 10. The pair of droplets of embodiment 9, wherein thesilicone is a polydimethysiloxane.

Embodiment 11. The pair of droplets of embodiment 10, wherein thesilicone is a tri-block copolymer comprising a 65-mer ofpolydimethylsiloxane capped at both ends with a 6 mer ofpoly(2-methyl-2-oxazoline).

Embodiment 12. The pair of droplets of any one of the precedingembodiments, wherein the transcription/translation extract is aeukaryotic extract.

Embodiment 13. The pair of droplets of embodiment 12, wherein theeukaryotic extract is a HeLa cell extract.

Embodiment 14. The pair of droplets of any one of embodiments 1 to 13,wherein the bilayer of the amphipathic molecules has a diameter in therange from about 30 μm to about 1000

Embodiment 15. The pair of droplets of any one of embodiment 1 to 14,wherein the amphipathic molecules are the same or different in the twodroplets.

Embodiment 16. The pair of droplets of any one of embodiment 1 to 15,wherein the first aqueous solution and the second aqueous solution arethe same or different.

Embodiment 17. The pair of droplets of any one of embodiments 1 to 16,wherein the first or second droplet contains a test compound, apolypeptide, a polymer, or a combination thereof.

Embodiment 18. The pair of droplets of any one of embodiments 1 to 17,wherein the encoded membrane polypeptide is synthesized within the firstdroplet and inserted into the bilayer.

Embodiment 19. The pair of droplets of any one of embodiments 1 to 18,wherein the bilayer is stable for at least about 30 minutes.

Embodiment 20. The pair of droplets of any one of embodiments 1 to 19,wherein the concentration of the polymer in the hydrophobic medium isgreater than about 0.05 g/L up to about 0.15 g/L.

Embodiment 21. The pair of droplets of any one of embodiments 1 to 20,wherein the polymer is present in the first or second aqueous solution.

Embodiment 22. The pair of droplets of any one of embodiments 1 to 21,wherein the membrane polypeptide is a eukaryotic polypeptide.

Embodiment 23. A system comprising a bilayer of amphipathic moleculesprovided at the interface between a first droplet of a first aqueoussolution in a hydrophobic medium, the first droplet comprising a layerof amphipathic molecules around the surface of the first aqueoussolution, and containing a transcription/translation extract and aheterologous polynucleotide encoding a membrane polypeptide; and asecond droplet of a second aqueous solution in the hydrophobic medium,the second droplet comprising a layer of amphipathic molecules aroundthe surface of the second aqueous solution; wherein the bilayer containsa polymer and a membrane polypeptide.

Embodiment 24. The system of embodiment 23, wherein the membranepolypeptide is a channel or a pore.

Embodiment 25. The system of embodiment 23 or 24, wherein thehydrophobic medium is oil.

Embodiment 26. The system of embodiment 25, wherein the oil is ahydrocarbon oil, a silicone oil, a fluorocarbon oil, or a combinationthereof.

Embodiment 27. The pair of droplets of any one of embodiments 23 to 26,wherein the amphipathic molecules are lipid molecules.

Embodiment 28. The system of any one of embodiments 23 to 27, whereineach droplet has a volume of at least about 20 pL and less than about1000 nL.

Embodiment 29. The system of any one of embodiments 23 to 28, whereinthe first or second droplet has a volume greater than or equal to 100nL.

Embodiment 30. The system of any one of embodiments 23 to 29, whereinthe first or second droplet has a volume less than or equal to 800 nL.

Embodiment 31. The system of any one of embodiments 23 to 30, whereinthe polymer is a silicone.

Embodiment 32. The system of embodiment 31, wherein the silicone is apolydimethysiloxane.

Embodiment 33. The system of embodiment 32, wherein the silicone is atri-block copolymer comprising a 65-mer of polydimethylsiloxane cappedat both ends with a 6 mer of poly(2-methyl-2-oxazoline).

Embodiment 34. The system of any one of embodiments 23 to 33, whereinthe transcription/translation extract is a eukaryotic extract.

Embodiment 35. The system of embodiment 34, wherein the eukaryoticextract is a HeLa cell extract.

Embodiment 36. The system of any one of embodiments 23 to 35, whereinthe bilayer of the amphipathic molecules has a diameter in the rangefrom about 30 μm to about 1000 μm.

Embodiment 37. The system of any one of embodiment 23 to 36, wherein theamphipathic molecules are the same or different in the two droplets.

Embodiment 38. The system of any one of embodiment 23 to 37, wherein thefirst aqueous solution and the second aqueous solution are the same ordifferent.

Embodiment 39. The system of any one of embodiments 23 to 38, whereinthe first or second droplet contains a test compound, a polypeptide, apolymer, or a combination thereof.

Embodiment 40. The system of any one of embodiments 23 to 39, whereinthe encoded membrane polypeptide is synthesized within the first dropletand inserted into the bilayer.

Embodiment 41. The system of any one of embodiments 23 to 40, whereinthe bilayer is stable for at least about 30 minutes.

Embodiment 42. The system of any one of embodiments 23 to 41, whereinthe concentration of the polymer in the hydrophobic medium is greaterthan about 0.05 g/L up to about 0.15 g/L.

Embodiment 43. The system of any one of embodiments 23 to 42, whereinthe polymer is present in the first or second aqueous solution.

Embodiment 44. The system of any one of embodiments 23 to 43, whereinthe membrane polypeptide is a eukaryotic polypeptide.

Embodiment 45. A method of forming a system comprising a bilayer ofamphiphilic molecules provided at the interface between two droplets,comprising: forming a first droplet of a first aqueous solution in thehydrophobic medium, the first droplet comprising a layer of amphipathicmolecules around the surface of the first aqueous solution, andcontaining a transcription/translation extract and a heterologouspolynucleotide encoding a membrane polypeptide; forming a second dropletof a second aqueous solution in the hydrophobic medium, the seconddroplet comprising a layer of amphipathic molecules around the surfaceof the second aqueous solution; bringing the droplets into contact withone another in the hydrophobic medium so that a bilayer of theamphipathic molecules is formed as an interface between the contactingdroplets; and incorporating a polymer into the bilayer.

Embodiment 46. The method of embodiment 45, further comprisingincubating the first droplet under conditions such that the encodedmembrane polypeptide is synthesized.

Embodiment 47. The method of embodiment 45 or 46, further comprisingincubating the contacted droplets such that the membrane polypeptide isinserted into the bilayer.

Embodiment 48. The method of any one of embodiments 45 to 47, whereinthe membrane polypeptide is a channel or a pore.

Embodiment 49. The method of any one of embodiments 45 to 48, whereinthe membrane polypeptide is a eukaryotic membrane polypeptide.

Embodiment 50. The method of any one of embodiments 45 to 49, furthercomprising moving the droplets when the droplets are in contact with oneanother to vary the area of the bilayer of the amphipathic molecules.

Embodiment 51. The method of any one of embodiments 45 to 50, whereinthe step of bringing droplets into contact with one another comprisesmoving one of the droplets into contact with the other droplet.

Embodiment 52. The method of embodiment 51, wherein the moved droplet isattached to an electrode.

Embodiment 53. The method of any one of embodiments 45 to 52, furthercomprising separating droplets which have been brought into contact withone another.

Embodiment 54. The method of any one of embodiments 45 to 53, whereinforming the first droplet or the second droplet comprises: (a) forming adroplet of aqueous solution in the hydrophobic medium; (b) before orafter step (a), providing the amphipathic molecules in the hydrophobicmedium; (c) after steps (a) and (b), incubating the droplet for a timesufficient for the layer of amphipathic molecules to form.

Embodiment 55. The method of any one of embodiments 45 to 53, whereinforming the first droplet or the second droplets comprises: forming adroplet of aqueous solution containing the amphipathic molecules in thehydrophobic medium; and incubating the droplet for a time sufficient forthe layer of amphipathic molecules to form.

Embodiment 56. A method of analyzing membrane polypeptide function,comprising contacting a test compound with the pair of droplets of anyone of embodiments 1 to 22 or the system of any one of embodiments 23 to45; and measuring a detectable signal from the system in the presenceand in the absence of the test compound.

Embodiment 57. The method of embodiment 56, further comprising bringingelectrodes into electrical contact with the droplets when the dropletsare in contact with one another and measuring an electrical signal usingthe electrodes.

Embodiment 58. The method of embodiment 56, wherein the detectablesignal is a signal from a chromophore.

Embodiment 59. The method of any one of embodiments 56 to 58, whereinthe test compound is a small molecule or a polypeptide.

Embodiment 60. A droplet of aqueous solution in a hydrophobic medium,the droplet comprising a layer of amphipathic molecules around thesurface of the aqueous solution, and containing atranscription/translation extract; and a heterologous polynucleotideencoding a membrane polypeptide and the hydrophobic medium or an aqueousphase containing a polymer capable of insertion into a bilayer of theamphipathic molecules.

Embodiment 61. The droplet of embodiment 60, wherein the membranepolypeptide is a channel or a pore.

Embodiment 62. The droplet of embodiment 60 or 61, wherein thehydrophobic medium is oil.

Embodiment 63. The droplet of embodiment 62, wherein the oil is ahydrocarbon oil, a silicone oil, a fluorocarbon oil, or a combinationthereof.

Embodiment 64. The droplet of any one of embodiments 60 to 63, whereinthe amphipathic molecules are lipid molecules.

Embodiment 65. The droplet of any one of embodiments 60 to 64, whereinthe droplet has a volume of at least about 20 pL and less than about1000 nL.

Embodiment 66. The droplet of any one of embodiments 60 to 65, whereinthe droplet has a volume greater than or equal to 100 nL.

Embodiment 67. The droplet of any one of embodiments 60 to 66, whereinthe droplet has a volume less than or equal to 800 nL.

Embodiment 68. The droplet of any one of embodiments 60 to 67, whereinthe polymer is a silicone.

Embodiment 69. The droplet of embodiment 68, wherein the silicone is apolydimethysiloxane.

Embodiment 70. The droplet of embodiment 69, wherein the silicone is atri-block copolymer comprising a 65-mer of polydimethylsiloxane cappedat both ends with a 6 mer of poly(2-methyl-2-oxazoline).

Embodiment 71. The droplet of any one of embodiments 60 to 70, whereinthe concentration of the polymer in the hydrophobic medium is greaterthan about 0.05 g/L up to about 0.15 g/L.

Embodiment 72. The droplet of any one of embodiments 60 to 71, whereinthe aqueous phase containing the polymer is the aqueous solution withinthe droplet.

Embodiment 73. The droplet of any one of embodiments 60 to 72, whereinthe transcription/translation extract is a eukaryotic extract.

Embodiment 74. The droplet of embodiment 73, wherein the eukaryoticextract is a HeLa cell extract.

Embodiment 75. The droplet of any one of embodiments 60 to 74, whereinthe membrane polypeptide is a eukaryotic polypeptide.

Embodiment 76. The method of any one of embodiment 45 to 55, whereinincorporating a polymer into the bilayer comprises contacting the firstor the second droplet with a lipid-polymer film on a surface of asubstrate in the hydrophobic medium such that a lipid-polymer monolayerforms on a surface of the first or second droplet.

Embodiment 77. The method of embodiment 56, further comprising coatingthe substrate with a lipid-polymer solution to form the lipid-polymerfilm on the surface of the substrate.

Embodiment 78. The method of embodiment 56 or 57, wherein contacting thefirst or the second droplet with the lipid-polymer film occurs prior tobringing the droplets into contact with one another in the hydrophobicmedium.

Embodiment 79. The method of any one of embodiments 56 to 58, whereinthe lipid in the lipid-polymer film comprises1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC),1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE), or a mixturethereof.

Embodiment 80. The method of any one of embodiments 57 to 59, whereinthe lipid-polymer solution comprises a mass ratio of1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC):1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE): polymer of20:5:0.5 to 20:5:5, preferably the mass ratio is 20:5:1.

Embodiment 81. The method of any one of embodiments 45-55, wherein thehydrophobic medium or an aqueous phase contains the polymer andincorporating the polymer into the bilayer comprises the polymerself-inserting into the bilayer.

Embodiment 82. The method of any one of embodiments 45 to 61, whereinthe polymer is a silicone.

Embodiment 83. The method of embodiment 62, wherein the silicone is apolydimethysiloxane.

Embodiment 84. The method of embodiment 63, wherein the silicone is atri-block copolymer comprising a 65-mer of polydimethylsiloxane cappedat both ends with a 6-mer of poly(2-methyl-2-oxazoline).

Embodiment 85. A system comprising a membrane separating first andsecond volumes of aqueous solution, the membrane comprising a lipidbilayer and an ion channel providing a passageway from one side of themembrane to the other, wherein the membrane contains 30% or less byweight of an amphiphilic polymer.

Embodiment 86. A system of embodiment 85 wherein the membrane comprises0.1% to 10% by weight of the amphiphilic polymer.

Embodiment 87. The system of embodiments 85 or 86, wherein theamphiphilic polymer is a diblock or triblock copolymer.

Embodiment 88. A system of any of embodiments 85 to 87 wherein the firstaqueous solution is a droplet in a hydrophobic medium, the dropletcomprising a layer of lipid around its surface.

Embodiment 89. A system of embodiment 88 wherein the second aqueoussolution is a droplet in a hydrophobic medium the droplet comprising alayer of lipid around its surface.

Embodiment 90. A system of embodiment 88 wherein the second aqueoussolution is a hydrophilic layer.

Embodiment 91. A system according to any of the preceding embodimentswherein the first and second aqueous solutions each contain electrodesto provide a potential difference across the membrane.

Embodiment 92. A system according to any of the preceding embodimentswherein the first and/or second aqueous solutions comprise anelectrolyte.

Embodiment 93. A system according to any of the preceding embodimentswherein the first and/or second aqueous solutions comprise a membranedestabilizing agent.

Embodiment 94. A system according to any of the preceding embodimentswherein the first and/or second aqueous solutions comprise atranscription/translation extract.

Embodiment 95. A system according to any of the preceding embodimentsfurther comprising one of more droplets, each droplet comprising a layerof lipid around its surface and forming an interconnected dropletnetwork, wherein a lipid bilayer is formed at each interface betweendroplets and wherein each lipid bilayer comprises an ion channel.

Embodiment 96. A system comprising a membrane separating first andsecond volumes of aqueous solution, the membrane comprising a lipidbilayer and an ion channel providing a passageway from one side of themembrane to the other, wherein the first and/or second volumes ofaqueous solution comprise a lipid bilayer destabilizing agent whereinthe membrane contains an amount of amphiphilic polymer effective forstabilization of the membrane.

Embodiment 97. A composition comprising a membrane comprising a lipidbilayer and an ion channel providing a passageway from one side of themembrane to the other, wherein the membrane contains 30% or less byweight of an amphiphilic polymer.

Embodiment 98. A composition comprises a pair of droplets in ahydrophobic medium, the pair of droplets comprises a first droplet of afirst aqueous solution in the hydrophobic medium, the first dropletcomprising a layer of lipid molecules around the surface of the firstaqueous solution; and a second droplet of a second aqueous solution inthe hydrophobic medium, the second droplet comprising a layer of lipidmolecules around the surface of the second aqueous solution; the firstdroplet and the second droplet being in contact with one another suchthat a bilayer of the lipid molecules is formed as an interfacetherebetween; wherein the bilayer comprises an amount of amphipathicpolymer effective for stabilization of the bilayer.

Embodiment 99. A composition comprising a droplet in a hydrophobicmedium containing an aqueous solution and a hydrophilic layer, thedroplet comprising a layer of lipid molecules around the surface of thefirst aqueous solution and the hydrophilic layer comprising a layer oflipid molecules on the surface of the second aqueous solution; the firstdroplet and the hydrophilic layer being in contact with one another suchthat a bilayer of the lipid molecules is formed as an interfacetherebetween; wherein the bilayer contains an amount of amphipathicpolymer effective for stabilization of the bilayer .

Embodiment 100. A composition of embodiments 98 or 99 wherein theaqueous solution or the hydrophilic layer comprises a lipid bilayerdestabilizing agent.

In general, the invention may alternately comprise, consist of, orconsist essentially of, any appropriate components herein disclosed. Theinvention may additionally, or alternatively, be formulated so as to bedevoid, or substantially free, of any components, materials,ingredients, adjuvants or species used in the prior art compositions orthat are otherwise not necessary to the achievement of the functionand/or objectives of the present invention. The endpoints of all rangesdirected to the same component or property are inclusive andindependently combinable (e.g., ranges of “less than or equal to 25 wt%, or 5 wt % to 20 wt %,” is inclusive of the endpoints and allintermediate values of the ranges of “5 wt % to 25 wt %,” etc.).Disclosure of a narrower range or more specific group in addition to abroader range is not a disclaimer of the broader range or larger group.“Combination” is inclusive of blends, mixtures, alloys, reactionproducts, and the like. Furthermore, the terms “first,” “second,” andthe like, herein do not denote any order, quantity, or importance, butrather are used to denote one element from another. The terms “a” and“an” and “the” herein do not denote a limitation of quantity, and are tobe construed to cover both the singular and the plural, unless otherwiseindicated herein or clearly contradicted by context. “Or” means“and/or.” The suffix “(s)” as used herein is intended to include boththe singular and the plural of the term that it modifies, therebyincluding one or more of that term (e.g., the film(s) includes one ormore films). Reference throughout the specification to “one embodiment”,“another embodiment”, “an embodiment”, and so forth, means that aparticular element (e.g., feature, structure, and/or characteristic)described in connection with the embodiment is included in at least oneembodiment described herein, and may or may not be present in otherembodiments. In addition, it is to be understood that the describedelements may be combined in any suitable manner in the variousembodiments.

The modifier “about” used in connection with a quantity is inclusive ofthe stated value and has the meaning dictated by the context (e.g.,includes the degree of error associated with measurement of theparticular quantity). The notation “±10%” means that the indicatedmeasurement can be from an amount that is minus 10% to an amount that isplus 10% of the stated value. The terms “front”, “back”, “bottom”,and/or “top” are used herein, unless otherwise noted, merely forconvenience of description, and are not limited to any one position orspatial orientation. “Optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where the event occurs andinstances where it does not. Unless defined otherwise, technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of skill in the art to which this invention belongs. A“combination” is inclusive of blends, mixtures, alloys, reactionproducts, and the like.

All cited patents, patent applications, and other references areincorporated herein by reference in their entirety. However, if a termin the present application contradicts or conflicts with a term in theincorporated reference, the term from the present application takesprecedence over the conflicting term from the incorporated reference

While particular embodiments have been described, alternatives,modifications, variations, improvements, and substantial equivalentsthat are or may be presently unforeseen may arise to applicants orothers skilled in the art. Accordingly, the appended claims as filed andas they may be amended are intended to embrace all such alternatives,modifications variations, improvements, and substantial equivalents.

1.-38. (canceled)
 39. A system comprising a bilayer of amphipathicmolecules provided at the interface between a first droplet of a firstaqueous solution in a hydrophobic medium, the first droplet comprising alayer of amphipathic molecules around the surface of the first aqueoussolution, and containing a transcription/translation extract and aheterologous polynucleotide encoding a membrane polypeptide; and asecond droplet of a second aqueous solution in the hydrophobic medium,the second droplet comprising a layer of amphipathic molecules aroundthe surface of the second aqueous solution; wherein the bilayer containsa polymer and a membrane polypeptide.
 40. The system of claim 39,wherein the membrane polypeptide is a channel or a pore.
 41. The systemof claim 39, wherein the hydrophobic medium is oil.
 42. (canceled) 43.The pair of droplets of claim 39, wherein the amphipathic molecules arelipid molecules.
 44. The system of claim 39, wherein each droplet has avolume of at least about 20 pL and less than about 1000 nL. 45.-46.(canceled)
 47. The system of claim 39, wherein the polymer is asilicone. 48.-49. (canceled)
 50. The system of claim 39, wherein thetranscription/translation extract is a eukaryotic extract. 51.-54.(canceled)
 55. The system of claim 39, wherein the first or seconddroplet contains a test compound, a polypeptide, a polymer, or acombination thereof.
 56. (canceled)
 57. The system of claim 39, whereinthe bilayer is stable for at least about 30 minutes.
 58. (canceled) 59.The system of claim 39, wherein the polymer is present in the first orsecond aqueous solution.
 60. The system of claim 39, wherein themembrane polypeptide is a eukaryotic polypeptide.
 61. A method offorming a system comprising a bilayer of amphiphilic molecules providedat the interface between two droplets, comprising: forming a firstdroplet of a first aqueous solution in a hydrophobic medium, the firstdroplet comprising a layer of amphipathic molecules around the surfaceof the first aqueous solution, and containing atranscription/translation extract and a heterologous polynucleotideencoding a membrane polypeptide; forming a second droplet of a secondaqueous solution in the hydrophobic medium, the second dropletcomprising a layer of amphipathic molecules around the surface of thesecond aqueous solution; bringing the droplets into contact with oneanother in the hydrophobic medium so that a bilayer of the amphipathicmolecules is formed as an interface between the contacting droplets; andincorporating a polymer into the bilayer.
 62. The method of claim 61,further comprising incubating the first droplet under conditions suchthat the encoded membrane polypeptide is synthesized.
 63. The method ofclaim 61, further comprising incubating the contacted droplets such thatthe membrane polypeptide is inserted into the bilayer.
 64. The method ofclaim 61, wherein the membrane polypeptide is a channel or a pore. 65.The method of claim 61, wherein the membrane polypeptide is a eukaryoticmembrane polypeptide. 66.-100. (canceled)